Failure to apply standard limit-of-detection or limit-of-quantitation criteria to specialized pro-resolving mediator analysis incorrectly characterizes their presence in biological samples

Specializedpro-resolvingmediators(SPM)derivedfromoxygenationof longchain polyunsaturated fatty acids(PUFA)wereoriginally described by Serhan and colleagues and have been proposed as mediators of in ﬂ ammation resolution. Families of SPM described in the literature include lipoxins, resolvins, maresins, protectins and their peptide conjugates.Gomezandco-authorsreportedthatlevelsofplasmaSPMfrom patients with early rheumatoid arthritis predict response to biologic therapy after 6 months. SPM were measured in this study using liquid chromatographytandemmassspectrometry(LC-MS/MS).Onreviewing the methods, supplementary analytical data, and the online peer review ﬁ le, we note serious concerns, regarding both analytical methods and experimental conclusions. Application of this ﬂ awed methodology to SPM analysis brings into question the very occurrence of many of these lipids in biological samples, their proposed impact on in ﬂ ammatory processes, and claims of their utility as biomarkers. In Gomez et al. 1 , the authors do not use signal-to-noise ratio (S/N) for determining limit of detection (LOD), instead they cite the

Signal/noise (S/N) criteria is the approved methodology set by the International Conference on Harmonisation (ICH), the European Pharmacopoeia (Ph.Eur), the International Organisation for Standardisation (ISO), the European Medicines Agency (EMA), the Food and Drug Administration (FDA), the United States Pharmacopeia (USP), the International Union of Pure and Applied Chemistry (IUPAC), and the World Health Organisation (WHO) [1][2][3][4][5][6][7][8][9][10] .While validated assays have recommended S/N of around 10 for LOQ, and 3:1 for LOD 1 , the practice of many academic research labs has been to use around 5:1 for LOQ.Consistent with this, the FDA requires that the analyte response at the LOQ should be ≥ 5 times the analyte response of the zero calibrator 11 .
The Serhan/Dalli method is described in more detail in two online protocols, which confirm the lack of S/N criteria being applied to set LOD/LOQ 12,13 .Worryingly, the integration of peaks with S/N <3 is actively encouraged in one protocol 13 .In a second protocol, the use of 2000 cps as baseline is recommended, and both describe how to confirm the presence of lipids using 6 MS/MS "diagnostic ions" 12,13 .

Supplementary Methods
Newly opened SPE cartridges (Waters, Sep-Pak C18, 6 mL capacity, 500 mg) were conditioned with 5 mL of methanol (Fischer, HPLC grade) followed by 10 mL of Ultrapure water (Cayman).5 mL of phosphate buffered saline was then loaded and columns washed with 10 mL of Ultrapure water.3 mL ethyl acetate (Sigma-Aldrich, LC-MS grade) was used to elute oxylipins.This was evaporated under vacuum and samples re-dissolved in methanol, before being analysed using LC-MS/MS as described.LC-MS/MS was performed on a Nexera liquid chromatography system (Nexera X2, Shimadzu) coupled to a 6500 QTrap mass spectrometer (AB Sciex).Liquid chromatography was performed at 45 °C using a Zorbax Eclipse Plus C18 (Agilent Technologies) reversed phase column (150 × 2.1 mm, 1.8 μm) at a flow rate of 0.5 mL/min over 22.5 min.Mobile phase A was (95 % HPLC water/5 % mobile phase B; v/v and 0.1 % acetic acid) and mobile phase B was acetonitrile/methanol (800 ml + 150 ml; and 0.1 % acetic acid).The following linear gradient for mobile phase B was applied: 30 % for 1 min, 30 -35 % from 1 to 4 min, 35 -67.5 % from 4 to 12.5 min, 67.5 -100 % from 12.5 to 17.5 min and held at 100 % for 3.5 min, followed by 1.5 min at initial condition for column reequilibration.Injection volume was 5 µL.Lipids were analyzed in monitoring (MRM) mode with scheduling (55s) for the baseline integration experiment.Ionization was performed using electrospray ionization in the negative ion mode with the following MS parameters: temperature 475 °C, N2 gas, GS1 60 psi, GS2 60 psi, curtain gas 35 psi, ESI voltage -4.5 kV.Cycle time was 0.4 s.For MS/MS analysis, enhanced product ion mode was used with dynamic fill time.Data were integrated using Analyst software.Data showing integrated windows are shown as screenshots, while MS/MS analysis was copied into PowerPoint for minimal processing (linewidths, font sizes only) with no alterations to chromatographic or MS/MS data.MS/MS is presented as profile or centroid as described in Figure Legends.Oxylipin standards were from Cayman Chemical.S/N ratio was manually calculated by measuring peak height (down to the midpoint of the noise) and noise height (full height across a clearly representative area of baseline), and dividing signal by noise.An example is shown here 14 .1. From a total of 55 chromatograms, only 16 appear to be usable with S/N >5, while another 5 have S/N >3 so are potentially detected but not quantifiable.Of the 16, 3 don't have good enough standard traces to confirm retention time.

Supplementary Table
1.For all standards, injected amounts were so low that the MS/MS spectra are extremely poor quality, comprising a significant level of background noise ions.Due to this, the spectra are not useful when matching to sample spectra 2. SPM are denoted by stereospecific names in the study, however reverse phase LC is unable to distinguish enantiomers.It is considered more accurate to denote lipids using annotation that accurately describes their level of annotation, e.g. the lipid being called PD1 or PDX can be called instead 10,17-dihydroxydocosahexaenoic acid 3.Only one chromatogram is shown for each lipid, thus it is impossible to determine the quality of the remaining data, and whether a lipid present in one sample is also present in other patient samples remains unknown.4. For several pairs of lipids, elution is only 0.1 min apart but it is claimed that both lipids are present, even though only one peak is seen in samples: PD1 and PDX, RvD3 and 17R-RvD3, RvD1 and 17R-RvD1, Mar1 and 7S,14S-diHDHA.

Supplementary Figure 1 .
Further examples where baseline noise integration generates signals higher than 2000 cps.Panel A. Example chromatogram from LTB3 standard analysed using LC-MS/MS as described in Methods.Three separate analyses of a methanol injection, in the region where LTB3 elutes showing the areas where the signal was integrated.Panel B. Example chromatogram from 8-HETE standard analysed using LC-MS/MS as described in Methods.Three separate analyses of a methanol injection, in the region where 8-HETE elutes showing the areas where the signal was integrated.Panel C. Example chromatogram from 15-HETE standard analysed using LC-MS/MS as described in Methods.Two separate analyses of a methanol injection, in the region where 15-HETE elutes showing the areas where the signal was integrated.Supplementary Figure 2. Examples of baseline noise integration and the presence of false "diagnostic" ions in MS/MS from extracted buffer blanks.Panel A. Example chromatogram from 5-HETE standard analysed using LC-MS/MS as described in Methods.Analysis of a methanol injection, in the region where 5-HETE elutes showing the areas where the signal was integrated.Panel B. Chromatogram, monitoring for RvD1 at m/z 375-215 in standard and blank.Panel C. MS/MS at 7.2-7.4min, where the RvD1 standard elutes, showing isolation and fragmentation of ion at m/z 375.Zoomed in regions of centroid spectrum showing background ions incorrectly identified as "diagnostic" ions for RvD1, as labelled by red arrows.Supplementary Figure 3.The presence of putative "diagnostic" ions in MS/MS from extracted buffer blanks Panel A. Chromatogram, monitoring for RvD5 at m/z 359-199 in standard and blank.Panel C. MS/MS at 9.9-10.1 min, where the RvD5 standard elutes, showing isolation and fragmentation of ion at m/z 359.Zoomed in regions of centroid spectrum affirming that background ions can be incorrectly identified as "diagnostic" ions for RvD5 (red arrows).